Dynein recruitment to nuclear pores activates apical nuclear migration and mitotic entry in brain progenitor cells.

Radial glial progenitors (RGPs) are elongated epithelial cells that give rise to neurons, glia, and adult stem cells during brain development. RGP nuclei migrate basally during G1, apically using cytoplasmic dynein during G2, and undergo mitosis at the ventricular surface. By live imaging of in utero electroporated rat brain, we find that two distinct G2-specific mechanisms for dynein nuclear pore recruitment are essential for apical nuclear migration. The "RanBP2-BicD2" and "Nup133-CENP-F" pathways act sequentially, with Nup133 or CENP-F RNAi arresting nuclei close to the ventricular surface in a premitotic state. Forced targeting of dynein to the nuclear envelope rescues nuclear migration and cell-cycle progression, demonstrating that apical nuclear migration is not simply correlated with cell-cycle progression from G2 to mitosis, but rather, is a required event. These results reveal that cell-cycle control of apical nuclear migration occurs by motor protein recruitment and identify a role for nucleus- and centrosome-associated forces in mitotic entry. PAPERCLIP:

Role of Nesprin-2 and RanBP2 in BICD2-associated brain developmental disorders.

Bicaudal D2 (BICD2) is responsible for recruiting cytoplasmic dynein to diverse forms of subcellular cargo for their intracellular transport. Mutations in the human BICD2 gene have been found to cause an autosomal dominant form of spinal muscular atrophy (SMA-LED2), and brain developmental defects. Whether and how the latter mutations are related to roles we and others have identified for BICD2 in brain development remains little understood. BICD2 interacts with the nucleoporin RanBP2 to recruit dynein to the nuclear envelope (NE) of Radial Glial Progenitor cells (RGPs) to mediate their well-known but mysterious cell-cycle-regulated interkinetic nuclear migration (INM) behavior, and their subsequent differentiation to form cortical neurons. We more recently found that BICD2 also mediates NE dynein recruitment in migrating post-mitotic neurons, though via a different interactor, Nesprin-2. Here, we report that Nesprin-2 and RanBP2 compete for BICD2-binding in vitro. To test the physiological implications of this behavior, we examined the effects of known BICD2 mutations using in vitro biochemical and in vivo electroporation-mediated brain developmental assays. We find a clear relationship between the ability of BICD2 to bind RanBP2 vs. Nesprin-2 in controlling of nuclear migration and neuronal migration behavior. We propose that mutually exclusive RanBP2-BICD2 vs. Nesprin-2-BICD2 interactions at the NE play successive, critical roles in INM behavior in RGPs and in post-mitotic neuronal migration and errors in these processes contribute to specific human brain malformations.

LIS1 and NudE induce a persistent dynein force-producing state.

Cytoplasmic dynein is responsible for many aspects of cellular and subcellular movement. LIS1, NudE, and NudEL are dynein interactors initially implicated in brain developmental disease but now known to be required in cell migration, nuclear, centrosomal, and microtubule transport, mitosis, and growth cone motility. Identification of a specific role for these proteins in cytoplasmic dynein motor regulation has remained elusive. We find that NudE stably recruits LIS1 to the dynein holoenzyme molecule, where LIS1 interacts with the motor domain during the prepowerstroke state of the dynein crossbridge cycle. NudE abrogates dynein force production, whereas LIS1 alone or with NudE induces a persistent-force dynein state that improves ensemble function of multiple dyneins for transport under high-load conditions. These results likely explain the requirement for LIS1 and NudE in the transport of nuclei, centrosomes, chromosomes, and the microtubule cytoskeleton as well as the particular sensitivity of migrating neurons to reduced LIS1 expression.

Glycogen synthase kinase 3 induces multilineage maturation of human pluripotent stem cell-derived lung progenitors in 3D culture.

Although strategies for directed differentiation of human pluripotent stem cells (hPSCs) into lung and airway have been established, terminal maturation of the cells remains a vexing problem. We show here that in collagen I 3D cultures in the absence of glycogen synthase kinase 3 (GSK3) inhibition, hPSC-derived lung progenitors (LPs) undergo multilineage maturation into proximal cells, type I alveolar epithelial cells and morphologically mature type II cells. Enhanced cell cycling, one of the signaling outputs of GSK3 inhibition, plays a role in the maturation-inhibiting effect of GSK3 inhibition. Using this model, we show NOTCH signaling induced a distal cell fate at the expense of a proximal and ciliated cell fate, whereas WNT signaling promoted a proximal club cell fate, thus implicating both signaling pathways in proximodistal specification in human lung development. These findings establish an approach to achieve multilineage maturation of lung and airway cells from hPSCs, demonstrate a pivotal role of GSK3 in the maturation of lung progenitors and provide novel insight into proximodistal specification during human lung development.

Nuclear migration in mammalian brain development.

During development of the mammalian brain, neural stem cells divide and give rise to adult stem cells, glia and neurons, which migrate to their final locations. Nuclear migration is an important feature of neural stem cell (radial glia progenitor) proliferation and subsequent postmitotic neuronal migration. Defects in nuclear migration contribute to severe neurodevelopmental disorders such as microcephaly and lissencephaly. In this review, we address the cellular and molecular mechanisms responsible for nuclear migration during the radial glia cell cycle and postmitotic neuronal migration, with a particular focus on the role of molecular motors and cytoskeleton dynamics in regulating nuclear behavior.

Role of kinesins in directed adenovirus transport and cytoplasmic exploration.

Many viruses, including adenovirus, exhibit bidirectional transport along microtubules following cell entry. Cytoplasmic dynein is responsible for microtubule minus end transport of adenovirus capsids after endosomal escape. However, the identity and roles of the opposing plus end-directed motor(s) remain unknown. We performed an RNAi screen of 38 kinesins, which implicated Kif5B (kinesin-1 family) and additional minor kinesins in adenovirus 5 (Ad5) capsid translocation. Kif5B RNAi markedly increased centrosome accumulation of incoming Ad5 capsids in human A549 pulmonary epithelial cells within the first 30 min post infection, an effect dramatically enhanced by blocking Ad5 nuclear pore targeting using leptomycin B. The Kif5B RNAi phenotype was rescued by expression of RNAi-resistant Kif5A, B, or C, and Kif4A. Kif5B RNAi also inhibited a novel form of microtubule-based "assisted-diffusion" behavior which was apparent between 30 and 60 min p.i. We found the major capsid protein penton base (PB) to recruit kinesin-1, distinct from the hexon role we previously identified for cytoplasmic dynein binding. We propose that adenovirus uses independently recruited kinesin and dynein for directed transport and for a more random microtubule-based assisted diffusion behavior to fully explore the cytoplasm before docking at the nucleus, a mechanism of potential importance for physiological cargoes as well.

Disentangling the molecular determinants for Cenp-F localization to nuclear pores and kinetochores.

Cenp-F is a multifaceted protein implicated in cancer and developmental pathologies. The Cenp-F C-terminal region contains overlapping binding sites for numerous proteins that contribute to its functions throughout the cell cycle. Here, we focus on the nuclear pore protein Nup133 that interacts with Cenp-F both at nuclear pores in prophase and at kinetochores in mitosis, and on the kinase Bub1, known to contribute to Cenp-F targeting to kinetochores. By combining in silico structural modeling and yeast two-hybrid assays, we generate an interaction model between a conserved helix within the Nup133 ß-propeller and a short leucine zipper-containing dimeric segment of Cenp-F. We thereby create mutants affecting the Nup133/Cenp-F interface and show that they prevent Cenp-F localization to the nuclear envelope, but not to kinetochores. Conversely, a point mutation within an adjacent leucine zipper affecting the kinetochore targeting of Cenp-F KT-core domain impairs its interaction with Bub1, but not with Nup133, identifying Bub1 as the direct KT-core binding partner of Cenp-F. Finally, we show that Cenp-E redundantly contributes together with Bub1 to the recruitment of Cenp-F to kinetochores.

Replication of early and recent Zika virus isolates throughout mouse brain development.

Fetal infection with Zika virus (ZIKV) can lead to congenital Zika virus syndrome (cZVS), which includes cortical malformations and microcephaly. The aspects of cortical development that are affected during virus infection are unknown. Using organotypic brain slice cultures generated from embryonic mice of various ages, sites of ZIKV replication including the neocortical proliferative zone and radial columns, as well as the developing midbrain, were identified. The infected radial units are surrounded by uninfected cells undergoing apoptosis, suggesting that programmed cell death may limit viral dissemination in the brain and may constrain virus-associated injury. Therefore, a critical aspect of ZIKV-induced neuropathology may be defined by death of uninfected cells. All ZIKV isolates assayed replicated efficiently in early and midgestation cultures, and two isolates examined replicated in late-gestation tissue. Alteration of neocortical cytoarchitecture, such as disruption of the highly elongated basal processes of the radial glial progenitor cells and impairment of postmitotic neuronal migration, were also observed. These data suggest that all lineages of ZIKV tested are neurotropic, and that ZIKV infection interferes with multiple aspects of neurodevelopment that contribute to the complexity of cZVS.

Conformational changes in the adenovirus hexon subunit responsible for regulating cytoplasmic dynein recruitment.

UNLABELLED: Virus capsids provide genome protection from environmental challenges but are also poised to execute a program of compositional and conformational changes to facilitate virion entry and infection. The most abundant adenovirus serotype 5 (AdV5) capsid protein, hexon, directly recruits the motor protein cytoplasmic dynein following virion entry. Dynein recruitment is crucial for capsid transport to the nucleus and requires the transient exposure of AdV5 hexon to low pH, presumably mimicking passage through the endosomal compartment. These results suggest a pH-dependent capsid modification during early infection. The changes to hexon structure controlling this behavior have not been explored. We report that hexon remains trimeric at low pH but undergoes more subtle conformational changes. These changes are indicated by increased sensitivities to SDS-mediated dissociation and dispase proteolysis. Both effects are reversed at neutral pH, as is dynein binding by low-pH-treated hexon. Dispase cleavage, which we find maps to a specific site within hypervariable region 1 (HVR1) of AdV5 hexon, has no apparent effect on virion entry but completely inhibits its transport to the nucleus. In addition, an AdV5 mutant containing HVR1 of AdV48 is unable to bind dynein and is strongly inhibited in the postentry transport step. These results reveal that conformational changes involving hexon HVR1 are the basis for a novel viral mechanism controlling capsid transport to the nucleus. IMPORTANCE: The adenovirus serotype 5 (AdV5) capsid protein hexon recruits the molecular motor protein cytoplasmic dynein in a pH-dependent manner, a function critical for efficient transport toward the nucleus and AdV5 infectivity. In this work, we describe how low-pH exposure induces reversible structural changes in AdV5 hexon and how these changes affect dynein binding. In addition, we identified a pH-sensitive dispase cleavage site in hexon HVR1, which depends on the same structural changes and furthermore regulates dynein recruitment and capsid redistribution in infected cells. These data provide the first evidence relating long-known but subtle pH-dependent structural changes in hexon to a more recently established essential but poorly understood role in virus transport. These results have broad implications for understanding virus infectivity in general, and our ability to block the recruitment mechanism has potential therapeutic implications as well.

Asymmetric centrosome inheritance maintains neural progenitors in the neocortex.

Asymmetric divisions of radial glia progenitors produce self-renewing radial glia and differentiating cells simultaneously in the ventricular zone (VZ) of the developing neocortex. Whereas differentiating cells leave the VZ to constitute the future neocortex, renewing radial glia progenitors stay in the VZ for subsequent divisions. The differential behaviour of progenitors and their differentiating progeny is essential for neocortical development; however, the mechanisms that ensure these behavioural differences are unclear. Here we show that asymmetric centrosome inheritance regulates the differential behaviour of renewing progenitors and their differentiating progeny in the embryonic mouse neocortex. Centrosome duplication in dividing radial glia progenitors generates a pair of centrosomes with differently aged mother centrioles. During peak phases of neurogenesis, the centrosome retaining the old mother centriole stays in the VZ and is preferentially inherited by radial glia progenitors, whereas the centrosome containing the new mother centriole mostly leaves the VZ and is largely associated with differentiating cells. Removal of ninein, a mature centriole-specific protein, disrupts the asymmetric segregation and inheritance of the centrosome and causes premature depletion of progenitors from the VZ. These results indicate that preferential inheritance of the centrosome with the mature older mother centriole is required for maintaining radial glia progenitors in the developing mammalian neocortex.

Novel dynein DYNC1H1 neck and motor domain mutations link distal spinal muscular atrophy and abnormal cortical development.

DYNC1H1 encodes the heavy chain of cytoplasmic dynein 1, a motor protein complex implicated in retrograde axonal transport, neuronal migration, and other intracellular motility functions. Mutations in DYNC1H1 have been described in autosomal-dominant Charcot-Marie-Tooth type 2 and in families with distal spinal muscular atrophy (SMA) predominantly affecting the legs (SMA-LED). Recently, defects of cytoplasmic dynein 1 were also associated with a form of mental retardation and neuronal migration disorders. Here, we describe two unrelated patients presenting a combined phenotype of congenital motor neuron disease associated with focal areas of cortical malformation. In each patient, we identified a novel de novo mutation in DYNC1H1: c.3581A>G (p.Gln1194Arg) in one case and c.9142G>A (p.Glu3048Lys) in the other. The mutations lie in different domains of the dynein heavy chain, and are deleterious to protein function as indicated by assays for Golgi recovery after nocodazole washout in patient fibroblasts. Our results expand the set of pathological mutations in DYNC1H1, reinforce the role of cytoplasmic dynein in disorders of neuronal migration, and provide evidence for a syndrome including spinal nerve degeneration and brain developmental problems.

The Role of Nde1 Phosphorylation in Interkinetic Nuclear Migration and Neural Migration During Cortical Development.

Nde1 is a cytoplasmic dynein regulatory protein with important roles in vertebrate brain development. One noteworthy function is in the nuclear oscillatory behavior in neural progenitor cells, the control and mechanism of which remain poorly understood. Nde1 contains multiple phosphorylation sites for the cell cycle-dependent protein kinase CDK1, though the function of these sites is not well understood. To test their role in brain development we expressed phosphorylation-state mutant forms of Nde1 in embryonic rat brains using in utero electroporation. We find that Nde1 T215 and T243 phosphomutants block apical interkinetic nuclear migration (INM) and, consequently, mitosis in radial glial progenitor cells. Another Nde1 phosphomutant at T246 also interfered with mitotic entry without affecting INM, suggesting a more direct role for Nde1 T246 in mitotic regulation. We also found that the Nde1 S214F mutation, which is associated with schizophrenia, inhibits Cdk5 phosphorylation at an adjacent residue which causes alterations in neuronal lamination. These results together identify important new roles for Nde1 phosphorylation in neocortical development and disease, and represent the first evidence for Nde1 phosphorylation roles in INM and neuronal lamination.

Molecular mechanism for recognition of the cargo adapter Rab6GTP by the dynein adapter BicD2.

Rab6 is a key modulator of protein secretion. The dynein adapter Bicaudal D2 (BicD2) recruits the motors cytoplasmic dynein and kinesin-1 to Rab6GTP-positive vesicles for transport; however, it is unknown how BicD2 recognizes Rab6. Here, we establish a structural model for recognition of Rab6GTP by BicD2, using structure prediction and mutagenesis. The binding site of BicD2 spans two regions of Rab6 that undergo structural changes upon the transition from the GDP- to GTP-bound state, and several hydrophobic interface residues are rearranged, explaining the increased affinity of the active GTP-bound state. Mutations of Rab6GTP that abolish binding to BicD2 also result in reduced co-migration of Rab6GTP/BicD2 in cells, validating our model. These mutations also severely diminished the motility of Rab6-positive vesicles in cells, highlighting the importance of the Rab6GTP/BicD2 interaction for overall motility of the multi-motor complex that contains both kinesin-1 and dynein. Our results provide insights into trafficking of secretory and Golgi-derived vesicles and will help devise therapies for diseases caused by BicD2 mutations, which selectively affect the affinity to Rab6 and other cargoes.

Development and application of in vivo molecular traps reveals that dynein light chain occupancy differentially affects dynein-mediated processes.

The ability to rapidly and specifically regulate protein activity combined with in vivo functional assays and/or imaging can provide unique insight into underlying molecular processes. Here we describe the application of chemically induced dimerization of FKBP to create nearly instantaneous high-affinity bivalent ligands capable of sequestering cellular targets from their endogenous partners. We demonstrate the specificity and efficacy of these inducible, dimeric "traps" for the dynein light chains LC8 (Dynll1) and TcTex1 (Dynlt1). Both light chains can simultaneously bind at adjacent sites of dynein intermediate chain at the base of the dynein motor complex, yet their specific function with respect to the dynein motor or other interacting proteins has been difficult to dissect. Using these traps in cultured mammalian cells, we observed that induction of dimerization of either the LC8 or TcTex1 trap rapidly disrupted early endosomal and lysosomal organization. Dimerization of either trap also disrupted Golgi organization, but at a substantially slower rate. Using either trap, the time course for disruption of each organelle was similar, suggesting a common regulatory mechanism. However, despite the essential role of dynein in cell division, neither trap had a discernable effect on mitotic progression. Taken together, these studies suggest that LC occupancy of the dynein motor complex directly affects some, but not all, dynein-mediated processes. Although the described traps offer a method for rapid inhibition of dynein function, the design principle can be extended to other molecular complexes for in vivo studies.

A two-kinesin mechanism controls neurogenesis in the developing brain.

During the course of brain development, Radial Glial Progenitor (RGP) cells give rise to most of the neurons required for a functional cortex. RGPs can undergo symmetric divisions, which result in RGP duplication, or asymmetric divisions, which result in one RGP as well as one to four neurons. The control of this balance is not fully understood, but must be closely regulated to produce the cells required for a functioning cortex, and to maintain the stem cell pool. In this study, we show that the balance between symmetric and asymmetric RGP divisions is in part regulated by the actions of two kinesins, Kif1A and Kif13B, which we find have opposing roles in neurogenesis through their action on the mitotic spindle in dividing RGPs. We find that Kif1A promotes neurogenesis, whereas Kif13B promotes symmetric, non-neurogenic divisions. Interestingly, the two kinesins are closely related in structure, and members of the same kinesin-3 subfamily, thus their opposing effects on spindle orientation appear to represent a novel mechanism for the regulation of neurogenesis.

NudC is required for interkinetic nuclear migration and neuronal migration during neocortical development.

NudC is a highly conserved protein necessary for cytoplasmic dynein-mediated nuclear migration in Aspergillus nidulans. NudC interacts genetically with Aspergillus NudF and physically with its mammalian orthologue Lis1, which is crucial for nuclear and neuronal migration during brain development. To test for related roles for NudC, we performed in utero electroporation into embryonic rat brain of cDNAs encoding shRNAs as well as wild-type and mutant forms of NudC. We show here that NudC, like Lis1, is required for neuronal migration during neocorticogenesis and we identify a specific role in apical nuclear migration in radial glial progenitor cells. These results identify a novel neuronal migration gene with a specific role in interkinetic nuclear migration, consistent with cytoplasmic dynein regulation.

Mutually exclusive cytoplasmic dynein regulation by NudE-Lis1 and dynactin.

Cytoplasmic dynein is responsible for a wide range of cellular roles. How this single motor protein performs so many functions has remained a major outstanding question for many years. Part of the answer is thought to lie in the diversity of dynein regulators, but how the effects of these factors are coordinated in vivo remains unexplored. We previously found NudE to bind dynein through its light chain 8 (LC8) and intermediate chain (IC) subunits (1), the latter of which also mediates the dynein-dynactin interaction (2). We report here that NudE and dynactin bind to a common region within the IC, and compete for this site. We find LC8 to bind to a novel sequence within NudE, without detectably affecting the dynein-NudE interaction. We further find that commonly used dynein inhibitory reagents have broad effects on the interaction of dynein with its regulatory factors. Together these results reveal an unanticipated mechanism for preventing dual regulation of individual dynein molecules, and identify the IC as a nexus for regulatory interactions within the dynein complex.

NudE and NudEL are required for mitotic progression and are involved in dynein recruitment to kinetochores.

NudE and NudEL are related proteins that interact with cytoplasmic dynein and LIS1. Their functional relationship and involvement in LIS1 and dynein regulation are not completely understood. We find that NudE and NudEL each localize to mitotic kinetochores before dynein, dynactin, ZW10, and LIS1 and exhibit additional temporal and spatial differences in distribution from the motor protein. Inhibition of NudE and NudEL caused metaphase arrest with misoriented chromosomes and defective microtubule attachment. Dynein and dynactin were both displaced from kinetochores by the injection of an anti-NudE/NudEL antibody. Dynein but not dynactin interacted with NudE surprisingly through the dynein intermediate and light chains but not the motor domain. Together, these results identify a common function for NudE and NudEL in mitotic progression and identify an alternative mechanism for dynein recruitment to and regulation at kinetochores.

Dynein at the cortex.

Cytoplasmic dynein is a minus end directed microtubule motor protein with numerous functions during interphase and mitosis. Recent evidence has identified several roles mediated by a fraction of cytoplasmic dynein associated with the cell cortex. So far, these include nuclear migration, mitotic spindle orientation, and cytoskeletal reorientation during wound healing, but others are likely. The possibility that a cortically bound form of dynein might represent its most ancient evolutionary state is discussed.

Role of the kinetochore/cell cycle checkpoint protein ZW10 in interphase cytoplasmic dynein function.

Zeste white 10 (ZW10) is a mitotic checkpoint protein and the anchor for cytoplasmic dynein at mitotic kinetochores, though it is expressed throughout the cell cycle. We find that ZW10 localizes to pericentriolar membranous structures during interphase and cosediments with Golgi membranes. Dominant-negative ZW10, anti-ZW10 antibody, and ZW10 RNA interference (RNAi) caused Golgi dispersal. ZW10 RNAi also dispersed endosomes and lysosomes. Live imaging of Golgi, endosomal, and lysosomal markers after reduced ZW10 expression showed a specific decrease in the frequency of minus end-directed movements. Golgi membrane-associated dynein was markedly decreased, suggesting a role for ZW10 in dynein cargo binding during interphase. We also find ZW10 enriched at the leading edge of migrating fibroblasts, suggesting that ZW10 serves as a general regulator of dynein function throughout the cell cycle.